Production of Protein Kinases in E. coli

Abstract

Recombinant protein expression is widely used to generate milligram quantities of protein kinases for crystallographic, enzymatic, or other biophysical assays in vitro. Expression in E. coli is fast, cheap, and reliable. Here I present a detailed protocol for the production of human Aurora-A kinase. I begin with transformation of a suitable plasmid into an expression strain of E. coli, followed by growth and harvesting of bacterial cell cultures. Finally, I describe the purification of Aurora-A to homogeneity using immobilized metal affinity and size exclusion chromatographies.

Appendices

Appendix 1

¤ (Main)

• 0.00 Base CV 5.027 {mL} HisTrap_FF_5_mL

• 0.00 Alarm_Pressure Enabled 1.5 {MPa} 0.00 {MPa}

• 0.00 Flow 3 {mL/min}

¤ 0.00 Block Wash_column

• (Wash_column)

• 0.00 Base SameAsMain

• 3 End_Block

¤ 0.00 Block Load_sample

• (Load_sample)

• 0.00 Base Volume

• 0.00 Fractionation 30 mm 45 {mL} FirstTube Volume

• 0.00 OutletValve F2

• 0.00 InjectionMark

• 0.00 InjectionValve Inject

• 0.00 AutozeroUV

• 60 InjectionValve Load

• 65.00 Hold_Until UV Less_Than 20 {mAU} 25 {base}

• 65.00 End_Block

¤ 0.00 Block Elute_sample

• (Elute_sample)

• 0.00 Base SameAsMain

• 0.00 Gradient 100 {%B} 0.00 {base}

• 0.00 Fractionation 18 mm 4 {mL} FirstTube Volume

• 5 End_Block

¤ 0.00 Block Back_to_A

• (Back_to_A)

• 0.00 Base SameAsMain

• 0.00 Gradient 0.0 {%B} 0.00 {base}

• 0.00 FractionationStop

• 2 End_Block

Appendix 2

¤ (Main)

• 0.00 Base Volume

• 0.00 Alarm_Pressure Enabled 1 {MPa} 0.00 {MPa}

• 0.00 Flow 1 {mL/min}

• 10 InjectionValve Inject

• 10 OutletValve F2

• 10 Fractionation 18 mm 2 {mL} FirstTube Volume

• 10 AutozeroUV

• 15 InjectionValve Load

• 109 FractionationStop

• 150.00 End_Method